PROJECT SUMMARY Vibrio cholerae causes the disease cholera, an important public health problem worldwide. V. cholerae?s ability to cause epidemics is tied to its dissemination and survival in aquatic habitats and its transmission to the human host. The pathogen?s ability to form biofilms (i.e., matrix-enclosed, surface-associated communities) is crucial for its survival in aquatic habitats between epidemics and is advantageous for host-to-host transmission during epidemics. The signaling nucleotide cyclic dimeric guanosine monophosphate (c-diGMP) is broadly conserved in bacteria and is a key regulator of biofilm formation. Understanding of how c-diGMP controls biofilm formation, which environmental signals modulate c-diGMP levels and biofilm formation, and consequences of c-diGMP signaling in V. cholerae infectivity, transmission and dissemination, is limited. These information gaps will addressed by focusing on two specific aims. 1) Analyze c-diGMP signaling pathways that control biofilm formation dynamics; and 2) Analyze activation of c-diGMP signaling pathways and their consequences in V. cholerae infection cycle. Under the first aim, the molecular mechanism(s) through which c-diGMP controls production of the type IVa MSHA pilus, the primary component of initial surface attachment will be determined using structural and biochemical approaches. The down-stream c-diGMP signaling pathways initiated upon surface attachment will be analyzed by employing microscopy-based community tracking methods to measure motility, division, and second messenger signal levels. The mechanism by which specific key c-diGMP signaling proteins act to control surface attachment and biofilm matrix production will be determined using combination of genetic and biochemical approaches. Under the second aim, the mechanism of activation of key c-diGMP signaling proteins will be determined using structural and ligand binding studies. The role of c-diGMP signaling in in vivo biofilm formation, in V. cholerae transmission and dissemination will also be investigated, using state- of-the-art imaging tools and novel c-diGMP sensors. The proposed work will greatly advance the understanding of how c-diGMP signaling operates, identify the inputs that influence c-diGMP production and degradation, and unveil the biological consequences of c-diGMP signaling. The proposed work promises molecular/mechanistic insights that will allow us to devise ways to control c-di-GMP signal transduction pathways governing motility and biofilm formation, ultimately providing targets for the development of inhibitors of V. cholerae transmission.